We are probing the substate binding site of various polysaccharide-metabolizing enzymes, especially porcine pancreatic alpha amylase and Bacillus macerans cyclodestrin transglucanase. Sparsely modified amyloses will be prepared, for example deoxy, fluorodeoxy, and unsaturated, where the degree of modification is less than 0.1. These materials will be used as amylase substrates, and products containing the modified glucose units will be isolated. Structure analysis of modified oligosaccharides will enable us to pinpoint the positions on the substrate binding site that are critical for either hydrogen bonding or specific ring conformation. We will synthesize oligosaccharide lactones to probe the left hand region of the binding site. If the lactone residue is bound primarily at the first subsite to the left of the catalytic site, as we expect, the effect of oligosaccharide length and chemical modification will enable us to characterize the binding site more accurately. With B. macerans enzyme will examine the specificity of the transfer reaction in which oligosaccharide of polysaccharide chains are coupled to radioactive glucose. By using glucosyl cyclodestrins as donors and radioactive glucose as an acceptor, we will obtain radioactive, branched oligosaccharides. From the structures of these oligosaccharides we can deduce the specificity of the enzyme substites as regards the effect of a bulky substituent at that particular position of the substrate.